The complete mitochondrial genome of Chibiraga houshuaii (Lepidoptera, Limacodidae) and its phylogenetic implications.

Chibiraga is a mall East Asian genus in the family Limacodidae (slug-moths). The latter includes many agricultural pests. Mitochondrial genome analysis is an important tool for studying insect molecular identification and phylogenetics. However, there are very few mitogenome sequences available for Limacodidae species, and none for the genus Chibiraga at all. To explore the mitogenome features of Chibiraga and verify its phylogenetic position, the complete mitogenome of Chibiraga houshuaii was sequenced and annotated. The complete 15,487 bp genome encoded 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (CR). Most of the PCGs had typical ATN start codons and terminated with TAA or a single T residue. UUA (Leu2), AUU (Ile), UUU (Phe), AUA (Met) and AAU (Asn) were the five most frequently used codons. All tRNAs were folded into cloverleaf secondary structure, except for trnS1, which lacked the DHU arm. Phylogenetic analyses within the superfamily Zygaenoidea were performed based on multiple datasets from mitochondrial genes. The results showed that the families Phaudidae, Limacodidae and Zygaenidae were respectively recovered as monophyly; C. houshuaii was clustered in a clade with nettle type larvae in Limacodidae.

Combinatorial and frequency properties of the ribosome ancestors.

BACKGROUND: The current ribosome has evolved from the primitive stages of life on Earth. Its function is to build proteins and on the basis of this role, we are looking for a universal common ancestor to the ribosome which could: i) present optimal combinatorial properties, and ii) have left vestiges in the current molecules composing the ribosome (rRNA or r-proteins) or helping in its construction and functioning. METHODS: Genomic public databases are used for finding the nucleotide sequences of rRNAs and mRNA of r-proteins and statistical calculations are performed on the occurrence in these genes of some pentamers belonging to the RNA proposed as optimal ribosome ancestor. RESULTS: After having exhibited a possible solution to the problem of an RNA capable of catalyzing peptide genesis, traces of this RNA are found in many rRNAs and mRNA of r-proteins, as well as in factors contributing to the construction of the current ribosome. CONCLUSIONS: The existence of an optimal primordial RNA whose function is to facilitate the creation of peptide bonds between amino acids may have contributed to accelerate the emergence of the first vital processes. Its traces should be found in many living species inside structures structurally and functionally close to the ribosome, which is already the case in the species studied in this article.

The Role of Ribosomal Proteins eL15 and eL36 in the Early Steps of Yeast 60S Ribosomal Subunit Assembly.

Ribosomal proteins have important roles in maintaining the structure and function of mature ribosomes, but they also drive crucial rearrangement reactions during ribosome biogenesis. The contribution of most, but not all, ribosomal proteins to ribosome synthesis has been previously analyzed in the yeast Saccharomyces cerevisiae. Herein, we characterize the role of yeast eL15 during 60S ribosomal subunit formation. In vivo depletion of eL15 results in a shortage of 60S subunits and the appearance of half-mer polysomes. This is likely due to defective processing of the 27SA3 to the 27SBS pre-rRNA and impaired subsequent processing of both forms of 27SB pre-rRNAs to mature 25S and 5.8S rRNAs. Indeed, eL15 depletion leads to the efficient turnover of the de novo formed 27S pre-rRNAs. Additionally, depletion of eL15 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we have analyzed the impact of depleting either eL15 or eL36 on the composition of early pre-60S particles, thereby revealing that the depletion of eL15 or eL36 not only affects each other's assembly into pre-60S particles but also that of neighboring ribosomal proteins, including eL8. These intermediates also lack most ribosome assembly factors required for 27SA3 and 27SB pre-rRNA processing, named A3- and B-factors, respectively. Importantly, our results recapitulate previous ones obtained upon eL8 depletion. We conclude that assembly of eL15, together with that of eL8 and eL36, is a prerequisite to shape domain I of 5.8S/25S rRNA within early pre-60S particles, through their binding to this rRNA domain and the recruitment of specific groups of assembly factors.

MRT-ModSeq - Rapid Detection of RNA Modifications with MarathonRT.

Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT-ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and modification type. As a proof of concept, we use the MRT fingerprints of well-studied rRNAs to implement a general workflow for detecting RNA modifications. MRT-ModSeq rapidly detects positions of diverse modifications across a RNA transcript, enabling assignment of m1acp3Y, m1A, m3U, m7G and 2'-OMe locations through mutation-rate filtering and machine learning. m1A sites in sparsely modified targets, such as MALAT1 and PRUNE1 could also be detected. MRT-ModSeq can be trained on natural and synthetic transcripts to expedite detection of diverse RNA modification subtypes across targets of interest.

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA.

Nucleophilic addition of bisulfite to pyrimidine bases has been known for a half century, and the reaction has been in use for at least a quarter of a century for identifying 5-methylcytidine in DNA. This account focuses on the chemistry of bisulfite with pseudouridine, an isomer of the RNA nucleoside uridine in which the uracil base is connected to C1' of ribose via C5 instead of N1. Pseudouridine, Ψ, is the most common nucleotide modification found in cellular RNA overall, in part due to its abundance in rRNAs and tRNAs. It has a stabilizing influence on RNA structure because N1 is now available for additional hydrogen bonding and because the heterocycle is slightly better at π stacking. The isomerization of U to Ψ in RNA strands is catalyzed by 13 different enzymes in humans and 11 in E. coli; some of these enzymes are implicated in disease states which is testament to the biological importance of pseudouridine in cells. Recently, pseudouridine came into the limelight as the key modification that, after N1 methylation, enables mRNA vaccines to be delivered efficiently into human tissue with minimal generation of a deleterious immunogenic response. Here we describe the bisulfite reaction with pseudouridine which gives rise to a chemical sequencing method to map the modified base in the epitranscriptome. Unlike the reaction with cytidine, the addition of bisulfite to Ψ leads irreversibly to form an adduct that is bypassed during cDNA synthesis by reverse transcriptases yielding a characteristic deletion signature. Although there were hints to the structure of the bisulfite adduct(s) 30 to 50 years ago, it took modern spectroscopic and computational methods to solve the mystery. Raman spectroscopy along with extensive NMR, ECD, and computational work led to the assignment of the major product as the (R) diastereomer of an oxygen adduct at C1' of a ring-opened pseudouridine. Mechanistically, this arose from a succession of conjugate addition, E2 elimination, and a [2,3] sigmatropic rearrangement, all of which are stereodefined reactions. A minor reaction with excess bisulfite led to the (S) isomer of a S-adducted SO3- group. Understanding structure and mechanism aided the design of a Ψ-specific sequencing reaction and guided attempts to improve the utility and specificity of the method. Separately, we have been investigating the use of nanopore direct RNA sequencing, a single-molecule method that directly analyzes RNA strands isolated from cells after end-ligation of adaptor sequences. By combining the electrical current and base-calling data from the nanopore with dwell-time analysis from the helicase employed to deliver RNA to the nanopore, we were able to map Ψ sites in nearly all sequence contexts. This analysis was employed to find Ψ residues in the SARS-CoV-2 vRNA, to analyze the sequence context effects of mRNA vaccine synthesis via in vitro transcription, and to evaluate the impact of stress on chemical modifications in the E. coli ribosome. Most recently, we found that bisulfite treatment of RNA leading to Ψ adducts could modulate the nanopore signal to help in mapping modifications of low occupancy.

Visualizing the nucleoplasmic maturation of human pre-60S ribosomal particles.

Eukaryotic ribosome assembly is a highly orchestrated process that involves over two hundred protein factors. After early assembly events on nascent rRNA in the nucleolus, pre-60S particles undergo continuous maturation steps in the nucleoplasm, and prepare for nuclear export. Here, we report eleven cryo-EM structures of the nuclear pre-60S particles isolated from human cells through epitope-tagged GNL2, at resolutions of 2.8-4.3 Å. These high-resolution snapshots provide fine details for several major structural remodeling events at a virtual temporal resolution. Two new human nuclear factors, L10K and C11orf98, were also identified. Comparative structural analyses reveal that many assembly factors act as successive place holders to control the timing of factor association/dissociation events. They display multi-phasic binding properties for different domains and generate complex binding inter-dependencies as a means to guide the rRNA maturation process towards its mature conformation. Overall, our data reveal that nuclear assembly of human pre-60S particles is generally hierarchical with short branch pathways, and a few factors display specific roles as rRNA chaperones by confining rRNA helices locally to facilitate their folding, such as the C-terminal domain of SDAD1.

Principles of human pre-60S biogenesis.

During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.

[The Structure, Expression, and Non-Canonical Functions of Human rDNA: The Role of Non-Coding Regions].

The genes coding for the rRNAs seem evolutionary conserved on the first glance, but astonish one with their variability in the structure and a variety of functions on closer examination. The non-coding parts of rDNA contain regulatory elements, protein binding sites, pseudogenes, repetitive sequences, and microRNA genes. Ribosomal intergenic spacers are not only in charge with the nucleolus morphology and functioning, namely, the rRNA expression and ribosome biogenesis, but also control nuclear chromatin formation thus mediating cell differentiation. The alterations in the expression of these non-coding regions of rDNA in response to environmental stimuli underlie the keen sense of a cell to various types of stressors. Malfunctioning of this process may result in a wide range of pathologies from oncology to neurodegenerative disease and mental illness. Here, we observe to-date materials on the structure and transcription of the ribosomal intergenic spacer in humans and its role in rRNA expression, in-born disease development, and cancer.

G·U base pairing motifs in long non-coding RNAs.

Long non-coding RNAs (lncRNAs) are recently-discovered transcripts involved in gene expression regulation and associated with diseases. Despite the unprecedented molecular complexity of these transcripts, recent studies of the secondary and tertiary structure of lncRNAs are starting to reveal the principles of lncRNA structural organization, with important functional implications. It therefore starts to be possible to analyze lncRNA structures systematically. Here, using a set of prototypical and medically-relevant lncRNAs of known secondary structure, we specifically catalogue the distribution and structural environment of one of the first-identified and most frequently occurring non-canonical Watson-Crick interactions, the G·U base pair. We compare the properties of G·U base pairs in our set of lncRNAs to those of the G·U base pairs in other well-characterized transcripts, like rRNAs, tRNAs, ribozymes, and riboswitches. Furthermore, we discuss how G·U base pairs in these targets participate in establishing interactions with proteins or miRNAs, and how they enable lncRNA tertiary folding by forming intramolecular or metal-ion interactions. Finally, by identifying highly-G·U-enriched regions of yet unknown function in our target lncRNAs, we provide a new rationale for future experimental investigation of these motifs, which will help obtain a more comprehensive understanding of lncRNA functions and molecular mechanisms in the future.

Structure of the actively translating plant 80S ribosome at 2.2 Å resolution.

In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing.

Genome-wide identification of Kanamycin B binding RNA in Escherichia coli.

BACKGROUND: The aminoglycosides are established antibiotics that inhibit bacterial protein synthesis by binding to ribosomal RNA. Additional non-antibiotic aminoglycoside cellular functions have also been identified through aminoglycoside interactions with cellular RNAs. The full extent, however, of genome-wide aminoglycoside RNA interactions in Escherichia coli has not been determined. Here, we report genome-wide identification and verification of the aminoglycoside Kanamycin B binding to Escherichia coli RNAs. Immobilized Kanamycin B beads in pull-down assays were used for transcriptome-profiling analysis (RNA-seq). RESULTS: Over two hundred Kanamycin B binding RNAs were identified. Functional classification analysis of the RNA sequence related genes revealed a wide range of cellular functions. Small RNA fragments (ncRNA, tRNA and rRNA) or small mRNA was used to verify the binding with Kanamycin B in vitro. Kanamycin B and ibsC mRNA was analysed by chemical probing. CONCLUSIONS: The results will provide biochemical evidence and understanding of potential extra-antibiotic cellular functions of aminoglycosides in Escherichia coli.

Structural basis for reduced ribosomal A-site fidelity in response to P-site codon-anticodon mismatches.

Rapid and accurate translation is essential in all organisms to produce properly folded and functional proteins. mRNA codons that define the protein-coding sequences are decoded by tRNAs on the ribosome in the aminoacyl (A) binding site. The mRNA codon and the tRNA anticodon interaction is extensively monitored by the ribosome to ensure accuracy in tRNA selection. While other polymerases that synthesize DNA and RNA can correct for misincorporations, the ribosome is unable to correct mistakes. Instead, when a misincorporation occurs, the mismatched tRNA-mRNA pair moves to the peptidyl (P) site and, from this location, causes a reduction in the fidelity at the A site, triggering post-peptidyl transfer quality control. This reduced fidelity allows for additional incorrect tRNAs to be accepted and for release factor 2 (RF2) to recognize sense codons, leading to hydrolysis of the aberrant peptide. Here, we present crystal structures of the ribosome containing a tRNALys in the P site with a U•U mismatch with the mRNA codon. We find that when the mismatch occurs in the second position of the P-site codon-anticodon interaction, the first nucleotide of the A-site codon flips from the mRNA path to engage highly conserved 16S rRNA nucleotide A1493 in the decoding center. We propose that this mRNA nucleotide mispositioning leads to reduced fidelity at the A site. Further, this state may provide an opportunity for RF2 to initiate premature termination before erroneous nascent chains disrupt the cellular proteome.

Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Complete mitochondrial genome of Cyclopelta obscura (Lepeletier & Serville, 1825) (Hemiptera: Pentatomoidea: Dinidoridae) and phylogenetic analysis of Pentatomoidea species.

Cyclopelta obscura is a crop pest, which mainly damages legumes, especially Robinia pseudoacacia and Cercis chinensis. In recent years, many mitochondrial and nuclear DNA sequences of C. obscura have been sequenced and used for phylogenetic inference. However, the complete mitogenome has not been reported yet and studies on the phylogenetic relationships within Dinidoridae are rare. In this study, we sequenced the mitogenome of C. obscura and conducted comparative mitogenomic analyses of seven Dinidoridae species based on several different factors. The length of the mitogenome is 15,426 bp, which includes 37 typical mitochondrial genes (13 protein-coding genes (PCGs), 22 tRNAs, and 2 rRNAs) and a control region (796 bp long), as well as 13 intergenic spacers and 8 overlapping regions. Most PCGs of C. obscura began with the classical start codon ATN, while cox1 and nad4l used TTG, and nad1 used GTG. The Dinidoridae mitogenomes are highly conserved in terms of nucleotide composition, the codon usage of PCGs, and the secondary structure of tRNA. Phylogenetic analysis based on four datasets with two methods recovered the Dinidoridae as a monophyletic group with strong support values. All results indicate that Dinidoridae formed a sister group to Tessaratomidae, and (Tessaratomidae + Dinidoridae) formed a sister group to Cydnidae in most of the phylogenetic trees. Additionally, seven species within the Dinidoridae, we observed the following relationship: (Eumenotes sp. + (Cyclopelta parva + C. obscura)) + ((Megymenum gracilicorne + Megymenum brevicorne) + (Coridius chinensis + Coridius brunneu)).

rRNA expansion segment 7 in eukaryotes: from Signature Fold to tentacles.

The ribosomal core is universally conserved across the tree of life. However, eukaryotic ribosomes contain diverse rRNA expansion segments (ESs) on their surfaces. Sites of ES insertions are predicted from sites of insertion of micro-ESs in archaea. Expansion segment 7 (ES7) is one of the most diverse regions of the ribosome, emanating from a short stem loop and ranging to over 750 nucleotides in mammals. We present secondary and full-atom 3D structures of ES7 from species spanning eukaryotic diversity. Our results are based on experimental 3D structures, the accretion model of ribosomal evolution, phylogenetic relationships, multiple sequence alignments, RNA folding algorithms and 3D modeling by RNAComposer. ES7 contains a distinct motif, the 'ES7 Signature Fold', which is generally invariant in 2D topology and 3D structure in all eukaryotic ribosomes. We establish a model in which ES7 developed over evolution through a series of elementary and recursive growth events. The data are sufficient to support an atomic-level accretion path for rRNA growth. The non-monophyletic distribution of some ES7 features across the phylogeny suggests acquisition via convergent processes. And finally, illustrating the power of our approach, we constructed the 2D and 3D structure of the entire LSU rRNA of Mus musculus.

Mechanisms and players of mitoribosomal biogenesis revealed in trypanosomatids.

Translation in mitochondria is mediated by mitochondrial ribosomes, or mitoribosomes, complex ribonucleoprotein machines with dual genetic origin. Mitoribosomes in trypanosomatid parasites diverged markedly from their bacterial ancestors and other eukaryotic lineages in terms of protein composition, rRNA content, and overall architecture, yet their core functional elements remained conserved. Recent cryo-electron microscopy studies provided atomic models of trypanosomatid large and small mitoribosomal subunits and their precursors, making these parasites the organisms with the best-understood biogenesis of mitoribosomes. The structures revealed molecular mechanisms and players involved in the assembly of mitoribosomes not only in the parasites, but also in eukaryotes in general.

Phylogeny and domain architecture of plant ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal RNA. Protein translation is inhibited by ribosome depurination; therefore, RIPs are generally considered toxic to cells. The expression of some RIPs is upregulated by biotic and abiotic stress, though the connection between RNA depurination and defense response is not well understood. Despite their prevalence in approximately one-third of flowering plant orders, our knowledge of RIPs stems primarily from biochemical analyses of individuals or genomics-scale analyses of small datasets from a limited number of species. Here, we performed an unbiased search for proteins with RIP domains and identified several-fold more RIPs than previously known - more than 800 from 120 species, many with novel associated domains and physicochemical characteristics. Based on protein domain configuration, we established 15 distinct groups, suggesting diverse functionality. Surprisingly, most of these RIPs lacked a signal peptide, indicating they may be localized to the nucleocytoplasm of cells, raising questions regarding their toxicity against conspecific ribosomes. Our phylogenetic analysis significantly extends previous models for RIP evolution in plants, predicting an original single-domain RIP that later evolved to acquire a signal peptide and different protein domains. We show that RIPs are distributed throughout 21 plant orders with many species maintaining genes for more than one RIP group. Our analyses provide the foundation for further characterization of these new RIP types, to understand how these enzymes function in plants.

Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation.

Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3' region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.

The epitranscriptome in ageing and stress resistance: A systematic review.

Modifications of RNA, collectively called the "epitranscriptome", might provide novel biomarkers and innovative targets for interventions in geroscience but are just beginning to be studied in the context of ageing and stress resistance. RNA modifications modulate gene expression by affecting translation initiation and speed, miRNA binding, RNA stability, and RNA degradation. Nonetheless, the precise underlying molecular mechanisms and physiological consequences of most alterations of the epitranscriptome are still only poorly understood. We here systematically review different types of modifications of rRNA, tRNA and mRNA, the methodology to analyze them, current challenges in the field, and human disease associations. Furthermore, we compiled evidence for a connection between individual enzymes, which install RNA modifications, and lifespan in yeast, worm and fly. We also included resistance to different stressors and competitive fitness as search criteria for genes potentially relevant to ageing. Promising candidates identified by this approach include RCM1/NSUN5, RRP8, and F33A8.4/ZCCHC4 that introduce base methylations in rRNA, the methyltransferases DNMT2 and TRM9/ALKBH8, as well as factors involved in the thiolation or A to I editing in tRNA, and finally the m6A machinery for mRNA.

Chemical Modifications of Ribosomal RNA.

Cellular RNAs in all three kingdoms of life are modified with diverse chemical modifications. These chemical modifications expand the topological repertoire of RNAs, and fine-tune their functions. Ribosomal RNA in yeast contains more than 100 chemically modified residues in the functionally crucial and evolutionary conserved regions. The chemical modifications in the rRNA are of three types-methylation of the ribose sugars at the C2-positionAbstract (Nm), isomerization of uridines to pseudouridines (Ψ), and base modifications such as (methylation (mN), acetylation (acN), and aminocarboxypropylation (acpN)). The modifications profile of the yeast rRNA has been recently completed, providing an excellent platform to analyze the function of these modifications in RNA metabolism and in cellular physiology. Remarkably, majority of the rRNA modifications and the enzymatic machineries discovered in yeast are highly conserved in eukaryotes including humans. Mutations in factors involved in rRNA modification are linked to several rare severe human diseases (e.g., X-linked Dyskeratosis congenita, the Bowen-Conradi syndrome and the William-Beuren disease). In this chapter, we summarize all rRNA modifications and the corresponding enzymatic machineries of the budding yeast.